Single Stranded Binding (SSB) proteins are essential in all organisms due to their central roles in DNA replication, recombination and repair. Our focus is on E. coli SSB protein which is the prototypical homotetrameric bacterial SSB. E. coli SSB protein plays a central role in genome maintenance by binding single stranded (ss)DNA and by directly interacting, primarily via its 4 intrinsically disordered C- terminal tails, with a least 14 other SSB interacting proteins (SIPs) to bring them to their sites of action on DNA. Yet, how SSB selects among these many proteins (specificity) and what mechanisms these proteins use to gain access to SSB-coated ssDNA are not understood. E. coli SSB protein possesses four DNA binding domains and can bind ssDNA in multiple binding modes that differ dramatically in their properties, in particular, the number of subunits interacting with ssDNA, which affects ssDNA wrapping around the tetramer, and its inter-tetramer ssDNA binding cooperativity. The different properties of the different SSB-ssDNA binding modes can regulate access of other proteins to the ssDNA. We have recently made the surprising discovery that highly cooperative binding of SSB to ssDNA is regulated by its four intrinsically disordered C-terminal tails and have proposed a model for how these tails promote cooperativity that will be tested in the current proposal. This discovery has also allowed us to make the first SSB variant that eliminates cooperative binding and we have shown that cells expressing this variant are defective in some DNA repair processes. We will also use computational and experimental (ensemble and single molecule) approaches to examine how the amino acid composition of the intrinsically disordered linker region of the C-terminal tails affects its conformational states an cooperativity. We have also developed novel approaches to make SSB variants that can selectively populate the different SSB-ssDNA binding modes. These SSB variants will be used to determine whether either of the major binding modes is dispensable in E. coli and what processes might be affected if SSB is restricted from accessing that particular mode. Ensemble thermodynamic approaches will also be used to investigate a novel allosteric effect of the SSB C-terminal tails on binding of at least one SIP to ssDNA. E. coli SSB is an essential bacterial protein and is involved in all DNA metabolic processes through its interactions with ssDNA and other proteins (SIPs). These SSB-SIP interactions provide a novel target for potentially novel antibiotics and our basic studies of EcoSSB and its C-terminal tails can facilitate the search and development of such novel antibiotics.